JP2618315B2 - Cell culture carrier and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a carrier for cell culture and a method for producing the same. That is, the present invention relates to a cell culture microcarrier used when culturing adhesion-dependent cells, and more specifically, to adhere adhesion-dependent animal cells to a microcarrier and perform high-density culture in a suspension state. It relates to a suitable cell culture microcarrier.

[0002]

2. Description of the Related Art Conventionally, a microcarrier culture method has been used as one of the animal cell culture methods. Conventional microcarriers include dextran, cellulose, gelatin,
Polyacrylamide, polystyrene, and the like are used as the material, and the shape is granular, and the particle diameter is 50 to 100.
0 μm particles. It is used as a carrier for attaching and culturing animal cells on the surface of the particles.

[0003] The above carrier is filled in a stirring tank together with a culture solution, floated, and animal cells are adhered to the surface of the carrier and cultured to be used for production of interferon, monoclonal antibodies, and various viruses.

[0004]

It has been considered that a conventional microcarrier has a spherical shape having excellent fluidity and mechanical strength. In order to increase the cell attachment area, a method of reducing the particle diameter as much as possible and a method of forming porous particles have been adopted. However, in any of the conventional methods, the conventional carrier has a great problem when used on an industrial scale because the microcarriers collide with each other by stirring the culture solution in the agitation tank and the adhered cells are greatly damaged by the collision. It became.

[0005]

An object of the present invention is to solve the above-mentioned problems and to provide a carrier having excellent adhesion and growth properties, wherein the carrier is easily suspended by stirring in a culture solution, and is extremely flexible and elastic. An object of the present invention is to provide a carrier having no resistance due to collision and to provide a method for producing such a carrier.

[0006]

According to the present invention, the sponge is made of natural sponge refined for the above purpose, and the size of the sponge is 5 times.
This was achieved by using a carrier for cell culture characterized in that the carrier had a pH of from 00 to 5000 and a pH of greater than 7 and a weak alkalinity of 8.5 or less when wetted with water.

Further, according to the present invention, the first step of refining the sponge and the second step of drying the sponge obtained in the first step to obtain fine or coarse particles are provided. When,
The present invention has been achieved by a method for producing a carrier for cell culture, comprising a third step of cleaning and activating the fine or coarse particles of sponge obtained in the second step.

The sponge used in the present invention is a natural animal sponge, preferably horny sponges among vulgaris sponges, and mainly belongs to the bathing sponge family. This sponge is collected, the internal organs are removed therefrom, and washed and dried (abbreviated as sponge) as a raw material.

[0009] Sponge is a marine animal, and I, Br, Cl,
Since S, Na, Mg, Fe, Ca, K, and the like are contained as impurities, scouring is performed as follows to completely remove these impurities.

As scouring methods, sulfuric acid, hydrochloric acid, nitric acid,
The solution is treated with a 1-5% solution of oxalic acid or the like at room temperature for 1-20 hours, and then treated with a 0.1% -0.5% solution of caustic potassium, caustic soda, ammonia, sodium carbonate, etc., for 30-40 times.
C. for 5 to 60 minutes to wash and dehydrate with water. Optionally, use a 0.1-0.5% solution of potassium permanganate to
After treating at 5 to 35 ° C. for 20 minutes to 1 hour, treatment may be performed at room temperature using a 1 to 5% solution of sulfuric acid, hydrochloric acid, nitric acid, and oxalic acid.

The sponge thus refined is dried, and after drying, crushed by a crusher. The degree of crushing is 500μ
It is made into a fine body having a length of ５5000 μ and a thickness of 10 to 30 μ with branches, or a coarse body having a sponge network structure and a particle diameter of 0.5 mm to 5 mm. In this case, the size of the carrier may be appropriately selected according to the capacity of the stirring tank and the type of cells. When the capacity of the stirring tank is large, the coarse particles are easier to handle than the fine particles.

Next, the surface stains of the sponge adhered at the time of crushing the sponge are washed with purified water and cleaned. After washing with purified water to improve cell adhesion,
7.5-8.5, a temperature of 35-45 ° C, 10-30
For a minute. Adjustment of PH is caustic potash,
Caustic soda, ammonia, and sodium carbonate may be used. After the cleaning activation treatment, dehydration drying is performed.

The sponge constituting the carrier of the present invention thus obtained has a size of 500 μm to 5000 μm, a pH of greater than 7 when wet with water, and a weak alkalinity of 8.5 or less. .

The sponge of the present invention is a sponge, and as a result of analyzing the amino acid composition, it has an amino acid composition similar to Koken atelocollagen or gelatin used for the surface treatment of the conventional microcarrier. From this, good results of cell culture could be expected, and good results were obtained as shown in Examples.

In addition, the adhesion and growth of cells are enhanced by setting the vicinity of the isoelectric point of the sponge protein by the activation treatment. Washing with a neutral to slightly acidic solution is not preferred because it deviates from the isoelectric point of the sponge protein and decreases the cell adhesion and proliferation.

Further, the sponge of the present invention is put into a culture solution, and
The sponge stably floated in the liquid when stirred at a stirring speed of 0 rpn, and gradually settled off when the stirring was stopped. This is extremely effective during culturing and recovery.

The carrier for cell culture of the present invention is sponge, and has the property of being flexible and elastic when wet with water. Therefore, since the medium is extremely flexible and elastic, there is no resistance due to collision and no damage to adherent cells.

Further, when used in combination with other microcapsules, it functions as a carrier and at the same time acts as a cushion for collision between carriers, which is effective in reducing damage.

[0019]

The present invention will be described below with reference to examples.

Example 1 A sponge was treated with a 2% sulfuric acid solution at room temperature for 8 hours and washed with water. Then 2g of caustic soda
The solution was treated at 35 ° C. for 15 minutes with the / l solution and washed with water. After centrifugal dehydration and drying at room temperature, it is crushed by
It was a fine body having a length of μ to 2000 μ and a branch and a thickness of about 20 μ. The fine particles were washed with purified water, and the solution adjusted to PH8 with caustic soda was subjected to a cleaning activation treatment at 40 ° C. for 10 minutes, centrifugally dehydrated and dried. The resulting sponge was wetted with water and measured to have a pH of 7.5.

100 ml of the carrier prepared as described above and PBS
(Phosphate buffered saline) 50 ml was put into a 500 ml spinner flask and sterilized at 110 ° C. for 20 minutes. Then D
DME containing 5% fetal bovine serum after washing with MEM medium (Dulbecco's Modified Eagles Medium)
M medium (200 ml) was added at 37 ° C., 95% air, 5% CO ₂
At equilibrium. Using CHO-Kl (Chinese hamster uterine ovary cells) as the cells, the seeding density was 2.0 ×
It was 10 ⁵ cell / ml. The cell density after 4 days was 1.6 × 1
It grew to 0 ⁶ cell / ml.

Example 2 A sponge was treated with a 3% solution of hydrochloric acid at room temperature for 10 hours, washed with water, and then treated with a 0.1% solution of potassium permanganate at 30 ° C. for 30 minutes. And then 15 g at 35 ° C. with 2 g / l sodium hydroxide solution.
Treated for a minute and washed with water. After centrifugal dehydration and drying at room temperature, the mixture was subjected to a crushing machine to obtain coarse particles having a sponge network structure with a particle diameter of 1 to 2 mm. The fine particles were washed with purified water and adjusted to a pH of 7.5 with sodium carbonate at 40 ° C. for 1 hour.
A cleaning activation treatment was performed for 0 minutes, followed by centrifugal dehydration and drying.
The resulting sponge was wetted with water and measured to have a pH of 7.5.

100 ml of the carrier prepared as described above and PBS
Put 50 ml into a 500 ml spinner flask,
For 20 minutes. Then, the plate was washed with DMEM medium, and 200 ml of DMEM medium containing 5% fetal bovine serum was added thereto.
5% air, equilibrated with 5% CO _2. CHO as a cell
The seeding density was 2.0 × 10 ⁵ cells / ml using -Kl. After 4 days, the cells had grown to a density of 1.4 × 10 ⁶ cells / ml.

Claims (2)

(57) [Claims] 1. A sponge made of scoured natural sponge, the size of which is 500 μm to 5000 μm.
A carrier for cell culture, wherein H has a weak alkaline property of greater than 7 and 8.5 or less. 2. A first step of refining the sponge, a second step of drying the sponge obtained in the first step to form fine or coarse particles, and a step of: The method for producing a carrier for cell culture according to claim 1, comprising a third step of cleaning and activating the fine or coarse particles of the obtained sponge.